Enzymatic method for preparing rebaudioside j

ABSTRACT

Provided is a method for preparing Rebaudioside J using an enzymatic method, comprising using rebaudioside A as a substrate, and making the substrate, in the presence of a glycosyl donor, react under the catalysis of a UDP-glycosyltransferase-containing recombinant cell and/or UDP-glycosyltransferase prepared therefrom to generate Rebaudioside J.

REFERENCE TO SEQUENCES LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing, (filename: 3711_9320001 SequenceListing.txt; size: 6,347 bytes; and date ofcreation: Jun. 6, 2022), filed herewith, is incorporated herein byreference in its entirety.

TECHNICAL FIELD

The present invention relates to a method for preparing Rebaudioside J,and in particular, relates to a biological method for preparingRebaudioside J.

BACKGROUND

Sweetening agents are a class of food additives that have wideapplications in the production of food, beverages, and candies. They maybe added in the food production process, or alternatively may be usedthrough appropriate dilution as a substitute for sucrose in householdbaking. Sweetening agents include natural sweetening agents, forexample, sucrose, high fructose corn syrup, honey, etc., and artificialsweetening agents, for example, aspartame, saccharine, etc. .Steviosides are a class of natural sweetening agents extracted from theplant Stevia rebaudiana, and are widely used in food products andbeverages at present. The extract of Stevia rebaudiana contains avariety of steviosides comprising rebaudioside. Naturally extractedsteviosides have great differences in ingredients across differentbatches, and need subsequent purification.

The content of Rebaudioside J found in the steviosides of the Stevialeaves does not exceed 0.5%; thus, it is extremely difficult to obtain aextract of Rebaudioside J with high purity using the conventionalmethod. Therefore, there are limited the in-depth studies ofRebaudioside J, and the commercial application of Rebaudioside J ishindered.

SUMMARY

The technical problem to be solved by the present invention is toovercome the defects in the prior art. The present invention achieves soby providing a method for preparing Rebaudioside J using an enzymaticmethod. With such a method, rebaudioside J product with high purity canbe produced at a lower cost and with a shorter production cycle.

The following technical solution is employed by the present invention tosolve the technical problem described above.

Provided is a method for preparing Rebaudioside J using an enzymaticmethod, wherein in the method, rebaudioside A is used as a substrate;and in the presence of a glycosyl donor, Rebaudioside J is produced bymeans of reaction under the catalysis of recombinant cells containingUDP-glycosyltransferase and/or UDP-glycosyltransferase preparedtherefrom. UDP-glycosyltransferase (i.e., uridinediphosphoglycosyltransferase), also referred to as UGT, is alreadywell-known.

Preferably, the glycosyl donor is a rhamnosyl donor.

More preferably, the rhamnosyl donor is a UDP-rhamnose.

Preferably, the UDP-glycosyltransferase is a UGT-B from Oryza sativa(rice).

Preferably, the amino acid sequence of UGT-B from Oryza sativa is atleast 60% consistent with Sequence 2 as shown in the Sequence Listing.

More preferably, the amino acid sequence of UGT-B from Oryza sativa isat least 70% consistent with Sequence 2 as shown in the SequenceListing.

Further, the amino acid sequence of UGT-B from Oryza sativa is at least80% consistent with Sequence 2 as shown in the Sequence Listing.

Furthermore, the amino acid sequence of UGT-B from Oryza sativa is atleast 90% consistent with Sequence 2 as shown in the Sequence Listing.

According to one example, the amino acid sequence of UGT-B from Oryzasativa is completely identical with Sequence 2 as shown in the SequenceListing.

According to the present invention,the reaction is carried out in anaqueous system at a temperature of 4-50° C. and a pH of 5.0 to 9.0.Preferably, the reaction is carried out in an aqueous system at atemperature of 35-45° C. and a pH of 7.5 to 8.5. More Preferably, thereaction is carried out at a temperature of below 40° C. and a pH ofbelow 8.0.

More Preferably, the reaction is carried out in a phosphate buffersolution.

More Preferably, the reaction system contains recombinant cells ofUDP-glycosyltransferase and a cell-permeating agent, and the reaction iscarried out in the presence of the cell-permeating agent.

Further, the cell-permeating agent is toluene, and the volume ratioconcentration of toluene in the reaction system is 1-3%. Furthermore,the volume ratio concentration of toluene is 2%.

More Preferably, all the raw materials used in the reaction are addedinto a reaction kettle to be uniformly mixed and then placed at a settemperature for reaction while stirring. After the reaction iscompleted, a Rebaudioside J product which can meet the requirements foruse can be obtained through purification-processing. A specificpurification method is through post-processing including resinisolation; and a Rebaudioside J product with a purity as high as 95% canbe obtained.

Preferably, the recombinant cell is a microbial cell.

More Preferably, the microorganism is Escherichia coli, Saccharomycescerevisiae, or Pichia pastoris.

By means of the foregoing technical solution, the present invention hasthe following advantages in comparison with the prior art:

The method of preparing Rebaudioside J using the enzymatic methodprovided by the present invention has important application values. Asthe substrate Rebaudioside A can be obtained in large quantities throughusing the enzymatic method, the production of Rebaudioside J is nolonger limited by the quantity of raw materials. The production cost isthus greatly reduced. It should also be considered that because of thelow content of Stevioside in the plant, and there are many Steviosideswith different structures, it is rather difficult to extract a productwith high purity. When compared with the prior art for extractingRebaudioside J from Stevia leaves, the present invention provides aproduct with a higher purity by adopting the enzymatic synthesis method,which will promote the research and application of novel SteviosideRebaudioside J.

DETAILED DESCRIPTION OF THE INVENTION

For the structural formulas of Rebaudioside A and Rebaudioside J, seeFormulas I and II respectively.

The main synthesis route of Rubadioside J as provided by the presentinvention is as follows:

The UGT-B adopted in the present invention may exist in the form oflyophilized enzyme powder or in the recombinant cells.

The method for obtaining the UGT-B is as follows:

a recombinant Escherichia coli (or the other microorganisms) expressionstrain of UGT-B is obtained by utilizing molecular cloning technique andgenetic engineering technique; then the recombinant Escherichia coli isfermented to obtain recombinant cells containing UGT-B, or to prepareand obtain lyophilized powder of UGT-B from the above noted recombinantcells.

Both the molecular cloning technique and the genetic engineeringtechnique described in the present invention are already well-known. Themolecular cloning technique may be found in Molecular Cloning: ALaboratory Manual(3rd Edition) (J. Sambrook, 2005).

The expression steps of the recombinant strain herein constructed byemploying genetic engineering technique are as follows:

(1) (according to Sequence 1 and Sequence 2 as shown in the SequenceListing) the required gene fragment is genetically synthesized, ligatedinto a pUC57 vector, while respectively adding NdeI and BamHI enzymecutting sites at the two ends;

(2) each gene fragment is inserted into the corresponding enzyme cuttingsite of the expression vector pET30a through double digestion andligation, so that each gene is placed under the control of T7 promoter;(3) the recombinant plasmid is transformed into Escherichia coli BL21(DE3); the expression of the target protein is induced by utilizingIPTG; and then the expression strains of the recombinant Escherichiacoli of UGT-B is obtained.

The steps for preparing the recombinant cells containing UGT-B and thelyophilized powder of UGT-B by utilizing the expression strains of therecombinant Escherichia coli containing UGT-B are as follows:

the recombinant Escherichia coli expression strains containing UGT-Bwere inoculated into 4 ml of liquid LB medium according to a proportionof 1%; shake cultivation was carried out at 37° C. (at 200 rpm)overnight; the substance cultivated overnight was taken and inoculatedinto 50m1 of liquid medium according to a proportion of 1%; shakecultivation was carried out at 37° C. (at 200 rpm) overnight until theOD₆₀₀ value reached 0.6-0.8; then IPTG with a final concentration of 0.4mM was added in at 20° C. for shake cultivation overnight. After theinduction is completed, the cells were collected through centrifugation(8,000 rpm, 10 min); then the cells were resuspended with 5 ml 2 mmol/Lof phosphate buffer liquid (pH7.0) , to obtain the recombinant cells;then the cells were ultrasonically disrupted in ice bath; the homogenatewas centrifuged (8,000 rpm, 10 min); and the supernatant was collectedand lyophilized for 24 h to obtain the lyophilized powder.

The present invention is further described in details in combinationwith specific examples.

Example 1: Preparation of the Recombinant Escherichia Coli CellsContaining UGT-B

According to Sequence 1 and Sequence 2, the UGT-B gene fragment wasgenetically synthesized, while respectively adding NdeI and BamHI enzymecutting sites at the two ends, and ligated to the pUC57 vector (producedby Suzhou Jin Wei Zhi Biotech. Co., Ltd.). The UGT gene segment wasenzyme cut with restriction endonucleases NdeI and BamHI; and then thesegments were recovered and purified; a T4 ligase was added to ligatethe segments into the corresponding enzyme cutting sites of pET30a, inorder to transform the BL21 (DE3) strains.

The UGT strains were inoculated into 4 ml of liquid LB medium accordingto a proportion of 1%; shake cultivation was carried out at 37° C. (at200 rpm) overnight; the substance cultivated overnight was taken andinoculated into 50 ml of liquid LB medium according to a proportion of1%; shake cultivation was carried out at 37° C. (at 200 rpm) overnightuntil the OD₆₀₀ value reached 0.6-0.8; then IPTG with a finalconcentration of 0.4 mM was added in at 20° C. for shake cultivationovernight. After the induction is completed, the cells were collectedthrough centrifugation (8,000 rpm, 10 min); and the collected cells wereresuspended with 5 ml 2 mol/L of phosphate buffer (pH 7.0) to obtain therecombinant cells containing UGT-B for catalysis.

Example 2 Preparation of the lyophilized Powder of UGT-B

The recombinant cells containing UGT-B prepared in Example 1 wereultrasonically disrupted in ice bath; the homogenate was centrifuged(8,000 rpm, 10 min); and the supernatant was collected and lyophilizedfor 24 h to obtain the lyophilized powder of UGT-B.

Example 3: Synthesis of Rebaudioside J under the Catalysis ofUDP-glycosyltransferase with Rebaudioside A as the Substrate

In this example, lyophilized powder of UGT-B prepared according to themethod of Example 2 was used for the catalysis and synthesis ofRebaudioside J.

1L 0.05 mol/L of phosphate buffer solution (pH8.0), 2 g of UDP Rhamnose,1 g of Rebaudioside A, 10 g of lyophilized powder of UGT-B weresequentially added into the reaction system, and placed into a waterbath at 40° C. after evenly mixing, for reaction for 24 h while stirringat 300 rpm. After the reaction is completed, 500 μl of the reactantsolution was added into anhydrous methanol of the equal volume foruniformly mixing; then it was centrifuged at 8,000 rpm for 10 min; and ahigh performance liquid chromatography was used to detect thesupernatant filtration membrane (chromatographic conditions: column:Agilent eclipse sb-C18 4.6×150 mm; detection wavelength: 210 nm; mobilephase: acetonitrile: deionized water =24%: 76%; flow rate: 1.0 mL/min;column temperature: 30° C.). The conversion rate of Rebaudioside A wasgreater than 90%. After the supernatant was purified by post-processingsuch as isolating by silica resin and crystallizing, 0.52g ofRebaudioside J was obtained, and the purity of which was greater than95%.

Example 4: Synthesis of Rebaudioside J under the Catalysis ofRecombinant Cells of UDP-glycosyltransferase with Rebaudioside A as theSubstrate

In this example, recombinant cells containing UGT-B prepared accordingto the method of Example 1 was used for the catalysis and synthesis ofRebaudioside J.

1L 0.05 mol/L of phosphate buffer solution (pH8.0) , 2 g of UDPRhamnose, 1 g of Rebaudioside A, 20 ml of toluene, 40% of UGT-B wholecells were sequentially added into the reaction system, and placed intoa water bath at 40° C. after uniformly mixing, to react for 24 h whilestirring at 300 rpm. After the reaction is completed, 500 μl of thereactant solution was taken, and the supernatant was added withanhydrous methanol of the equal volume for uniformly mixing; then it wascentrifuged at 8,000 rpm for 10 min; and a high performance liquidchromatography was used to detect the supernatant filtration membrane(chromatographic conditions: column: Agilent eclipse sb-C18 4.6×150 mm;detection wavelength: 210 nm; mobile phase: acetonitrile: deionizedwater=24%: 76%; flow rate: 1.0 mL/min; column temperature: 30° C.). Theconversion rate of Rebaudioside A was greater than 90%. After thesupernatant was purified by post-processing such as isolating by silicaresin and crystallizing, 0.49 g of Rebaudioside J was obtained, and thepurity of which was greater than 95%.

The above-described examples are merely for the illustration of thetechnical concept and features of the present invention. The object ofproviding examples is only to allow those skilled in the art tounderstand the present invention and implement it accordingly; the scopeof the present invention is not limited thereto. Any equivalentvariations or modifications derived from the essence of the presentinvention shall fall within the protection scope of the presentinvention.

1-15. (canceled)
 16. An enzymatic method for preparing Rebaudioside J,the method comprising reacting Rebaudioside A with a rhamnosyl donor ina reaction system comprising: recombinant cells comprising aUDP-glycosyltransferase, a UDP-glycosyltransferase prepared therefrom,or both the recombinant cells comprising the UDP-glycosyltransferase andthe UDP-glycosyltransferase prepared therefrom, wherein theUDP-glycosyltransferase comprises an amino acid sequence having at least80% sequence identity to SEQ ID NO:
 2. 17. The method of claim 16,wherein the UDP-glycosyltransferase comprises an amino acid sequencehaving at least 90% sequence identity to SEQ ID NO:
 2. 18. The method ofclaim 17, wherein Rebaudioside A is converted to Rebaudioside J at aconversion rate greater than 90%.
 19. The method of claim 17, whereinthe rhamnosyl donor is a UDP-rhamnose.
 20. The method of claim 17,wherein the UDP-glycosyltransferase is a UGT-B from Oryza sativa. 21.The method of claim 17, wherein the reaction system is aqueous and has atemperature of 35-45° C. and a pH of 7.5 to 8.5.
 22. The method of claim21, wherein the reaction system comprises a phosphate buffer solution.23. The method of claim 21, wherein the reaction system furthercomprises a cell-permeabilizing agent.
 24. The method of claim 23,wherein the cell-permeabilizing agent is toluene and wherein the toluenehas a concentration by volume of 1-3%.
 25. The method of claim 17,wherein the recombinant cell is a cell of a microorganism.
 26. Themethod of claim 25, wherein the microorganism is Escherichia coli,Saccharomyces cerevisiae, or Pichia pastoris.
 27. The method of claim17, further comprising purifying the Rebaudioside J via resin isolation.28. The method of claim 27, wherein the Rebaudioside J purified viaresin isolation has a purity greater than 95%.
 29. A recombinant cellcomprising a UDP-glycosyltransferase, wherein theUDP-glycosyltransferase comprises an amino acid sequence having at least80% sequence identity to SEQ ID NO:
 2. 30. The recombinant cell of claim29, wherein the UDP-glycosyltransferase comprises an amino acid sequencehaving at least 90% sequence identity to SEQ ID NO:
 2. 31. Therecombinant cell of claim 30, wherein the recombinant cell is selectedfrom the group consisting ofEscherichia coli, Saccaromyces cerevisiae,and Pichia pastoris.
 32. The recombinant cell of claim 30, wherein theUDP-glycosyltransferase is a uridine diphosphoglycosyltransferase-B(UGT-B) from Oryza sativa.
 33. A method of preparing a lyophilizedpowder of UDP-glycosyltransferase, comprising disrupting the recombinantcell of claim 30, obtaining a cell homogenate, centrifuging the cellhomogenate to obtain a supernatant, and lyophilizing the supernatant.34. The method of claim 33, wherein the recombinant cell is selectedfrom the group consisting of Escherichia coli, Saccaromyces cerevisiae,and Pichia pastoris.
 35. The method of claim 33, wherein theUDP-glycosyltransferase is a uridine diphosphoglycosyltransferase-B(UGT-B) from Oryza sativa.